1. Field of the Invention
The present invention relates generally to microscope calibration and more particularly to the calibration of fluorescence detection across a field of view of a fluorescence microscope.
2. Background Information
The fluorescence microscope has become a valuable tool in biology and biomedical science, as well as in material science and other fields, due to its unique detection abilities. Such detection abilities are a result of the microscope's method of operation. When an organic or inorganic specimen (a target) is exposed to intense light (termed excitation light) it will generally emit light (termed emission light), in the physical phenomenon of fluorescence. Emission light typically is very faint, having only an intensity between a hundred thousandth and a millionth the intensity of the excitation light. A fluorescence microscope operates by separating the faint emission light from any reflected excitation light, and thereafter measuring the emission light to identify structures in the target.
In modern microscopy, the excitation light is commonly generated by epi-florescence illumination, where light of a defined wavelength is applied to the target (an instrument operating in this manner is termed an epi-florescence microscope). By using a defined wavelength, excitation light may be more easily separated from the emission light. Further, in addition to measuring the natural fluorescence of the target (the auto-fluorescence), it is common to use fluorescent dies (fluorochromes) that attach to specific structures of the target and emit light of known wavelengths when excited. In such a manner, specific features of the target may be studied, for example the presence of particular molecules may be detected.
Fluorescence microscopy may be better understood through reference to FIG. 1, which is a schematic diagram of an exemplary fluorescence microscope 100. An illumination source 110 creates high intensity light that may be multispectral or limited to certain wavelengths. The illumination source is commonly an arc-discharge lamp, such as a mercury or xenon burner. Alternatively, the illumination source may be an integrated assembly of Light Emitting Diodes (LEDs) such as the Luxeon® Star LEDs available from Lumileds Lighting, LLC of San Jose, Calif. In other configurations, the illumination source may be a laser light source.
Light from the illumination source generally passes through one or more collimating lenses 120 that convert the illumination light into a collimated beam, whose rays are parallel to each other. An excitation filer 130, following the collimating lenses 120, selectively transmits a particular wavelength, or narrow band of wavelengths, and blocks light of other wavelengths. The transmitted wavelengths (now termed excitation light) enter an optical block 150 of the microscope 100. The excitation light generally enters the optical block 150 perpendicular to the optical axis of the microscope 100.
In the optical block 150, the excitation light is reflected by a dichromatic beam-splitting mirror 140 (called a dichroic). The dichroic 140 is tilted at a 45-degree angle with respect to the incoming excitation light and thus reflects the light downward into an objective optical system 160 toward a target 190. Typically, a shutter mechanism 195 is placed before the target, so that exposure time may be regulated. In a typical fluorescence microscope, the target is illuminated across a field of view of about ½ to 1 square centimeters. When the target is illuminated by the excitation light, it fluoresces (either through autofluorescence or due to the application of fluorochromes). Thereafter, florescence emission (emission light) from the illuminated target travels upwards and is collected by the objective optical system 160, now serving in its usual image-forming function. In addition to the emission light, large amounts of excitation light are reflected from the target and re-enter the objective optical system 160. The emission light and the excitation light reflected from the target are separated by the dichroic, which selectively reflects wavelengths associated with the excitation light back towards the illumination source, where they are dissipated. The emission light passes through the dichroic 140 to an emission filter 170 that suppresses residual excitation light. The emission light then enters an optical eyepiece assembly (not shown) for viewing by an operator, or enters an image sensing device 180, such as a Charge-Coupled Device (CCD) camera. The image sensing device 180 contains an array of sensors, each of which measures (samples) the light at a different point in the field of view to form a pixel (i.e. a small discrete component of a digital image). Digital images are typically composed of a large number of pixels, and image sensing devices often describe images using several million pixels (megapixels).
To fully exploit the capabilities of a florescence microscope, an adequate calibration technique is needed. Unfortunately, calibration of a florescence microscope is different from, and more demanding than, calibration of a conventional optical microscope, and is still an outstanding problem. Conventional optical microscopes are generally adequately calibrated by standardizing qualities such as resolution, contrast, depth of field, and distortion. To that end, calibration targets have been employed consisting of printed or vapor deposited patterns on substrates, such as glass or plastic slides.
Yet fluorescence microscopes also require calibration of fluorescence detection, permitting emission light intensity to be measured accurately across a field of view. That is, calibration is required so that the measured intensity differences across a field of view are due to differences in the target and not “irregularities” in the fluorescence microscope itself. Known calibration techniques have proven inadequate for this type of calibration.
Unwanted variation in emission light is primarily caused by variation in excitation light intensity, which can vary on the order of 3:1 between highs and lows across the field of view. Further unwanted variation is introduced by irregularities in the lenses of the optical path of the microscope. Such variation is generally not apparent when an image is observed by the human eye, as the human eye is a poor detector of intensity deviations. Yet, when an image is electronically captured and quantified, such variation is clearly apparent. Without an acceptable degree of calibration, “quantified fluorescent microscopy” (i.e. the assignment of numerical values to image characteristics) proves impracticable. Uncalibrated results may not readily be compared between instruments, or even between differing regions of a field of view of the same instrument.
One application adversely affected by a lack of calibration of fluorescence detection is the examination of biochips and microarrays, where target materials are affixed to a substrate in a 2-dimensional array of spots. Such microarrays are used in bioassay methodologies where a number of biologically identical spots are laid down upon the substrate, each being an independent assay. Statistical analysis across the array is typically used to increase accuracy. Yet, if there is considerable unwanted variation in emission light intensity, spots in differing location may not adequately be compared. Thus, without adequate calibration, the utility of this technique is reduced.
As stated above, existing techniques for calibrating fluorescence microscopes have not adequately addressed calibration of fluorescence detection across a field of view. With most prior techniques, fluorescence detection may not be calibrated to greater than a relative accuracy of 10%. As such, existing techniques are generally unsuitable for quantified fluorescent microscopy, where accuracies of 1% or greater are desired.
One existing calibration technique involves layering organic fluorescent material about 30 microns (micrometers) in thickness upon a non-fluorescent glass substrate, such as synthetic quartz. Since fluorescent material emits light throughout its thickness, for such a technique to be capable of intensity calibration to 1% accuracy, the thickness of the fluorescent material would need to be controlled to within 30 nanometers (nm). This is impractical given present manufacturing technology and economic constraints, and, accordingly, much lesser accuracy levels must be accepted.
Another existing technique for calibrating a fluorescence microscope involves a substrate of fluorescent glass on which a very thin patterned metal layer (such as a nickel layer) is deposited. While this general technique has been advantageously employed in the calibration of image resolution, it offers little precision in calibrating fluorescence detection. The glass substrate's emissions vary significantly throughout the field of view. Such variations are due to both thickness variations and non-uniformities in the glass's composition. Accuracy may be improved somewhat by depositing a thin layer of Kapton® film (available from DuPont High Performance Materials Inc.) on an opaque cover on the glass substrate. This improved technique is described in U.S. Pat. No. 6,472,671 to Montagu, issued on Oct. 29th, 2002, which is incorporated herein by reference in its entirety. Use of a Kapton® film may allow thickness variations in the fluorescence source to be reduced to 0.1 micron. Yet this is still insufficient for high accuracy calibration.
A wide variety of other calibration techniques involving calibration targets, surfaces, and coatings are commercially available. Yet, absent the use of exotic and cost-prohibitive materials and manufacturing methods, these techniques are unable to achieve a relative accuracy in emission intensity measurement greater than about 10%. What is needed is a relatively simple and inexpensive system and method for calibrating a fluorescence microscope that allows one to calibrate fluorescence detection across the entire field of view of a microscope to an acceptable level of accuracy. Such a system and method would be highly advantageous to the field of quantified fluorescence microscopy.